Abstract

SummarySeveral ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific “affimer” reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.

Highlights

  • Polyubiquitination of proteins is an important posttranslational modification that can lead to a variety of cellular outcomes

  • Once a Ub chain is formed, it is recognized by Ub-binding domains (UBDs) (Husnjak and Dikic, 2012), and this can occur in a linkage-specific manner

  • Selective UBDs for five out of the eight linkage types are known to date (Swatek and Komander, 2016; Yau and Rape, 2016)

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Summary

Introduction

Polyubiquitination of proteins is an important posttranslational modification that can lead to a variety of cellular outcomes. The best-studied role of ubiquitination is as a proteasomal degradation tag, but ubiquitin (Ub) has many non-degradative roles (Swatek and Komander, 2016; Yau and Rape, 2016). This versatility originates in part from the ability of Ub to form distinct polyUb chains. Selective UBDs for five out of the eight linkage types are known to date (Swatek and Komander, 2016; Yau and Rape, 2016). Deubiquitinases (DUBs) disassemble the Ub chains, and some cleave chains with high linkage selectivity (Clague et al, 2013; Mevissen and Komander, 2017)

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